Identification of different gene expressions between diffuse- and intestinal-type spheroid-forming gastric cancer cells

Background: Three-dimensional in vitro spheroid models are unique because they are considered for enrichment of specific cell populations with self-renewal ability. In this study, we explored the different mechanisms of gastric cancer spheroid-forming cells according to the Lauren classification. Methods: We isolated and enriched cells with self-renewal ability using spheroid-forming methods from gastric cancer cell lines. The expression of candidate target genes was investigated using western blot and qRT-PCR analysis. Lentiviral shRNA knockdown of target gene expression was performed and the effects on spheroid, colony forming, and tumorigenic ability were analyzed. Results: The SNU-638, SNU-484, MKN-28, and NCI-N87 successfully formed spheroid from single cell and enriched for self-renewal ability from 11 gastric cancer cell lines, including diffuse and intestinal types. The expression of SOX2 and E-cadherin increased in spheroid-forming cells in a diffuse-type cell line (SNU-638 and SNU-484), but not in the intestinal type (MKN-28 and NCI-N87). In contrast, ERBB3 expression was only increased in intestinal-type spheroid cells. The depletion of each candidate target gene expression suppressed self-renewal ability to grow as spheroids and colonies in a soft agar assay. In particular, down-regulated ERBB3 in the intestinal-type cell lines inhibited tumor growth in a mouse xenograft model. We found that high ERBB3 gene expression correlates with decreased survival in the intestinal type of gastric cancer. Conclusions: Our results suggest that diffuse- and intestinal-type spheroid-forming cells express genes differently. Our data suggest that these candidate genes from spheroid-forming cells can be used in applications in targeted therapy.